Bladder cancer detection using microsatellite analysis in paired buccal swab and urine samples

ABSTRACT

Methods are described for the efficient and accurate detection of bladder cancer. In particular, the method utilizes microsatellite analysis of bladder cancer markers to determine the presence of loss of heterozygosity or microsatellite instability using matched buccal swab and urine samples from a patient. In some cancer marker panels, detected loss of heterozygosity or microsatellite instability in two markers can be indicative of bladder cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 17/383,291 filed Jul. 22, 2021, which is acontinuation application of U.S. patent application Ser. No. 16/060,620filed Jun. 8, 2018, which is the National Stage Application ofInternational Patent Application No. PCT/US2016/058229 filed Oct. 21,2016, which claims priority to U.S. Provisional Patent Application No.62/264,504, filed Dec. 8, 2015, the entire disclosures of which arehereby incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

Disclosed herein are compositions and methods useful for detection ofbladder cancer.

BACKGROUND OF THE INVENTION

Bladder cancer is the fourth and seventh most common malignancy inAmerican men and women, respectively. Annually, 60,000 and 18,000 newcases of bladder cancer are diagnosed in American men and women,respectively. About 75% of patients have a superficial form of thedisease, and 15% of these patients are at risk of the diseaseprogressing to an invasive form. About 70% of patients with thesuperficial form of the disease experience disease recurrence within 10years, with the majority of recurrences being detected within two yearsof the initial diagnoses. For these reason, patients with thesuperficial form of the disease require monitoring for diseaseprogression and recurrence.

Currently, the standard of care for bladder cancer surveillance includesa cystoscopy and urinary cytology every three months for two yearsfollowed by annual cystoscopies, urinary cytologies, and radiographicevaluations of the upper urinary tract. However, this surveillanceapproach not optimal as the sensitivity and specificity for cytology isonly 25-50% and 90-100%, respectively. While sensitivity of cystoscopyis higher (90-100%), it is an expensive procedure that requiresintricate instrumentation and a sterile environment. And because of itsinvasive nature, the procedure has inherent risks of injury,complications, and infection.

Thus, there is a need for methods of detecting bladder cancer that areboth sensitive and specific for the disease and that have less risk ofcomplications compared to other detection methods. The disclosed methodsand kits directed to these and other important needs.

SUMMARY OF THE INVENTION

To address, inter alia, the unmet needs described above, disclosedherein are methods for detecting loss of heterozygosity in a subjectcomprising amplifying a set of microsatellite markers from a bladdersample and a matched control sample to produce a set of amplificationproducts, wherein the set of markers comprise FGA, D9S747, MBP, D9S162,TH01, IFN-A, D21S1245, and D20S48; detecting the amplification products;comparing the amplification products from the bladder sample and theamplification products from the matched control sample; and determiningif loss of heterozygosity is present in any of the markers in thebladder sample.

Methods are also provided for detecting bladder cancer in a subjectcomprising amplifying a set of microsatellite markers from a bladdersample and a control sample using the primers described herein;detecting the reaction product; and determining if more than one of themicrosatellite markers exhibits loss of heterozygosity or microsatelliteinstability in the bladder sample, wherein in at least twomicrosatellite markers exhibiting loss of heterozygosity or instabilityis indicative of bladder cancer.

Also disclosed herein are kits comprising primers having specificnucleic acid sequences and packaging for said primers.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the disclosed methods and kits, there are shownin the drawings exemplary embodiments of the methods and kits; however,the methods and kits are not limited to the specific embodimentsdisclosed. In the drawings:

FIG. 1 illustrates LOH at the IFN-A locus;

FIG. 2 illustrates the detection of addional alleles in the D9S171locus;

FIG. 3 illustrates an artifact detected in the D16S476 locus;

FIG. 4 provides an electropherogram of showing LOH at an STR markerlocated at 9q32;

FIG. 5 provides a summary of characteristics and associated samplenumbers for study subjects having bladder cancer;

FIG. 6A shows the MSA profile for control subject N1;

FIG. 6B shows the MSA profile for control subject N2;

FIG. 6C shows the MSA profile for control subject N3;

FIG. 6D shows the MSA profile for control subject N4;

FIG. 6E shows the MSA profile for control subject N5;

FIG. 6F shows the MSA profile for bladder cancer subject C1;

FIG. 6G shows the MSA profile for bladder cancer subject C2;

FIG. 6H shows the MSA profile for bladder cancer subject C3;

FIG. 6I shows the MSA profile for bladder cancer subject C4; and

FIG. 6J shows the MSA profile for bladder cancer subject C5.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed methods and kits may be understood more readily byreference to the following detailed description taken in connection withthe accompanying figures, which form a part of this disclosure. It is tobe understood that the disclosed methods and kits are not limited to thespecific methods and kits described and/or shown herein, and that theterminology used herein is for the purpose of describing particularembodiments by way of example only and is not intended to be limiting ofthe claimed methods and kits.

Unless specifically stated otherwise, any description as to a possiblemechanism or mode of action or reason for improvement is meant to beillustrative only, and the disclosed methods and kits are not to beconstrained by the correctness or incorrectness of any such suggestedmechanism or mode of action or reason for improvement.

Throughout this text, the descriptions refer to compositions and methodsof using said compositions. Where the disclosure describes or claims afeature or embodiment associated with a composition, such a feature orembodiment is equally applicable to the methods of using saidcomposition. Likewise, where the disclosure describes or claims afeature or embodiment associated with a method of using a composition,such a feature or embodiment is equally applicable to the composition.

When a range of values is expressed, another embodiment includes fromthe one particular value and/or to the other particular value. Further,reference to values stated in ranges include each and every value withinthat range. All ranges are inclusive and combinable. When values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment.Reference to a particular numerical value includes at least thatparticular value, unless the context clearly dictates otherwise.

It is to be appreciated that certain features of the disclosed methodsand kits which are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features of the disclosed methods andkits that are, for brevity, described in the context of a singleembodiment, may also be provided separately or in any subcombination.

As used herein, the singular forms “a,” “an,” and “the” include theplural.

Various terms relating to aspects of the description are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinitions provided herein.

As used herein, “microsatellite,” (used interchangeably with “simplesequence repeat” (SSR) and “short tandem repeat” (STR)) refers tosegments of nucleic acids having contiguous repeats of a base unitconsisting of two to six base pairs. Microsatellites are generally100-400 base pairs in length. Thus, if the base unit of themicrosatellite is 4 base pairs, there will be about 25 to 100 contiguousrepeats of the base unit.

“Microsatellite instability” refers changes that occur to the nucleicacid sequence of microsatellites in a cell when the mismatch repair(MMR) is not functioning properly. The characteristic repeat units of amicrosatellite are prone to errors caused by failure of DNA polymerases'inherent proof-reading capability to correct mismatches. Accumulation ofinsertions or deletions of nucleobases can lead to frameshift mutationsthat can have deleterious effects on protein expression. The MMR systemcan be viewed as a fail-safe system because those mismatch errorsundetected by the DNA polymerase are corrected by the MMR. Mutations inthe genes encoding the MMR system result in lack of surveillance ofmicrosatellite replication and instability can result. Microsatelliteinstability occurring in tumor suppressor genes has been associated withcancer phenotypes.

The term “loss of heterozygosity,” or LOH, refers to loss of a segmentof DNA, most likely due to replication error in one chromosomal strandbut not in the other. In some cancers, LOH can be particularlyproblematic if the lost genetic material encodes all or part of a tumorsuppressor protein, cellular reproduction machinery, or other proteinsnecessary for the stable reproduction of cells. Although the remainingcopy may be sufficient to prevent any deleterious effects caused by thelost allele, LOH can result in—or at least contribute to—a cancerphenotype if the remaining copy is insufficient to prevent deleteriouseffects or if the remaining copy is mutagenized such that the encodedprotein does not function as a wildtype protein. LOH at a microsatelliteallele may be associated with cancer because the remainingmicrosatellite allele, still susceptible to mutation—and especially soin a cell lacking a fully functional MMR system—may acquire furthermutations or variations that result in a non-functioning protein. If thenonfunctioning protein is a tumor suppressor, there is a heightened riskof a cancer phenotype.

In addition to the approximately 80,000 new cases of bladder cancerdiagnosed each year, approximately 12,000 men and 4,500 women dies fromthe disease annually. Because of the prevalence of the disease in theUnited States and abroad, much research has been undertaken to identifygenetic components that may be contributing or otherwise indicative ofthe disease. This research has identified numerous tumor markersassociated—in some capacity—with the disease, and in the past decade,several tumor markers have been incorporated into in vitro diagnosticassays for bladder tumor detection. Currently, BTA™, BTA™ Stat, FDP™,and NMP-22™ have been cleared by the Food and Drug Administration (FDA)as adjuncts to cystology and cytology, and these tests are usuallyantibody-mediated detection of tumor markers. Sensitivity andselectivity profiles for these assays suggest that although these markerdetection methods may augment contemporary invasive detectionprocedures, their sensitivity and selectivity profiles suggest that theycannot be relied on without a companion diagnostic (Halachmi et al.,Molecular Urology, 1(4):309-314 (1997); Han and Schoenberg, UrologicOncology, 5(3):87-92 (2000)). For example, sensitivity of the BLA Stattest, depending on the stage of disease, can be as low as 51% and as lowas 42% for stage 1 cancers.

The present invention, in contrast, is a method of detecting bladdercancer without the need for invasive techniques or a combinatorialapproach to diagnosis. More specifically, the present invention detectsLOH or MSI in bladder cells by analyzing a specific set ofmicrosatellite markers from a bladder specimen, wherein LOH detected attwo markers in a bladder sample is indicative of disease. The disease,in some embodiments of the present invention, is bladder cancer.

While no invasive techniques are necessary to obtain test specimens, theinvention is not limited to only non-invasively derived samples. Samplesobtained from biopsy, surgery, and even autopsy can be used to detectbladder cancer using the presently described techniques. In some aspectsof the present invention, the bladder sample can be a urine sample, andin other aspects, the bladder sample can be a bladder biopsy sample. Insome embodiments of the present invention, a matched control sample isblood, blood serum, buccal cells, hair follicle, saliva, sebum, skin,sweat, or tears. In some aspects, the matched control sample is buccalcells.

Microsatellites can be used as markers to detect, diagnose, and/or studydiseases when the microsatellites associated with a particular disease.The present invention discloses a method of detecting changes in a panelof microsatellite markers. In order to detect these markers, nucleicacid from a bladder specimen must be isolated. Amplifying of amicrosatellite allows for one skilled in the art to detect the presenceof, absence of, or changes to the microsatellite. The microsatellite isflanked by sequences that can be used to design primers for use inamplifying the microsatellite. Thus, in some aspects of the presentinvention, each marker is amplified using a pair of primers. Table 1recites the primers used for each marker amplified in one embodiment ofthe present invention.

Amplification of nucleic acids is accomplished using any techniquecapable of amplifying nucleic acids, such as isothermal amplificationreactions, which amplify nucleic acids at a substantially isothermaltemperature, or polymerase chain reactions (PCR), which utilize multiplecycles of different temperatures to denaturate template nucleic acids,anneal primers, and extend a nascent nucleic acid strand. PCR reactionsinclude, but are not limited to, traditional PCR, real time (RT) PCR,and qualitative PCR (qPCR). Any amplification protocol that allows forthe discrimination of amplified nucleic acids based on size iscontemplated in the present invention. In one embodiment of the presentinvention, a microsatellite marker is amplified from nucleic acidsobtained from a urine sample and from a buccal sample. The resultingamplified nucleic acids will have an expected length, and any derivationfrom that expected length, including the complete absence of anamplified product, may signal microsatellite instability or loss ofheterozygosity.

One embodiment of the present invention provides a method for detectingLOH in a subject comprising amplifying microsatellite markers from abladder sample and a matched control sample to produce amplificationproducts, wherein the markers comprise FGA, D9S747, MBP, D9S162, TH01,IFN-A, D21S1245, and D20S48; detecting the amplification products;comparing the amplification products from the bladder sample and theamplification products from the matched control sample; and determiningif loss of heterozygosity is present in any of the markers in thebladder sample.

Amplified nucleic acids can be resolved using molecular methods known inthe art including, but not limited to, agarose gel electrophoresis,capillary electrophoresis, and high pressure liquid chromatography. Whencomparing a single amplified product from a bladder sample and a singleamplified product from a control sample, agarose gel electrophoresisallows for a visual determination if the samples possess the markernucleic acid. Capillary electrophoresis also allows for the resolutionof differently sized amplification product in an automated fashion, andthis method is especially well suited for resolving multipleamplification products in a single capillary.

Some aspects of the present invention provide for amplifying a pluralityof markers in a single amplification reaction. Such an approach requiresdetermining the proper reaction conditions (e.g., primer combinations,ionic strength, temperature conditions, etc.). Because some markerscannot be efficiently amplified, unambiguously identified afteramplification, or are present in limiting amounts, more than oneamplification reaction may be required. Thus, amplification of themarkers comprises at least two multiplex amplification reactions in someembodiments. In some aspects, amplification of the markers comprisesthree multiplex amplification reactions. In some embodiments of thepresent invention in which the amplification of markers comprises threeamplification reactions, the markers to be amplified in one multiplexamplification reaction comprise FGA and D9S747. In some embodiments, themarkers to be amplified in one multiplex amplification reaction compriseD9162, MBP, IFN-A, and THO1, or any subcombination thereof. For example,one multiplex amplification reaction may include only D9162 and MBP. Insome embodiments, the markers to be amplified in one multiplexamplification reaction comprise D21S1245 and D20S48.

While multiplex amplification reactions may be an efficient mechanism todetect LOH or MSI of a marker, each marker may be amplifiedindividually. The individual amplification reaction products may then becombined prior analysis for LOH or MSI. Each amplification product mayalso be analyzed individually to determine if LOH or MSI is present.

Table 1 lists attributes of three multiplex reactions (MP1, MP2, andMP3) including the marker name; the chromosome (Chr) upon which eachmarker resides; the short tandem repeat sequence for each marker; theforward and reverse primers; and the dyes conjugated to the forwardprimer. For example, the STR for marker D4S243 is (ATAG)_(n), where“ATAG” is the repeat unit and “n” is the number of times the unit isrepeated. D4S243 is located on chromosome 4, and the forward primer islabeled with 6-FAM.

TABLE 1 Multi- Forward Primer Reverse Primer Plex Marker Chr. STRSequence (5′-3′) Sequence (5′-3′) MP1 D4S243 4 (ATAG)n 6FAM-TAGGAGCCTGTGGTCCTGTT TCAGTCTCTCTTTCTCCTTGCA (SEQ ID NO: 2)(SEQ ID NO: 1) FGA 4 (TTTC)n VIC- CTTCTCAGATCCTCTGACACTCGGACATCTTAACTGGCATTCATG (SEQ ID NO: 4) G (SEQ ID NO: 3) D9S747 9 (GATA)nVIC- CAGGCTCTCAAAATATGAACAAAAT GCCATTATTGACTCTGGAAAAG (SEQ ID NO: 6)AC (SEQ ID NO: 5) D17S654 17 (CA)n NED- GAGCAGAATGAGAGGCCAAGACCTAGGCCATGTTCACAGC (SEQ ID NO: 8) (SEQ ID NO: 7) D17S695 16 (AAAG)nPET- TTTGTTGTTGTTCATTGACTTCAGTC CTGGGCAACAAGAGCAAAAT (SEQ ID NO: 10)(SEQ ID NO: 9) MP2 D9S162 9 (CA)n NED- TCCCACAACAAATCTCCTCACGCAACCATTTATGTGGTTAGGG (SEQ ID NO: 12) (SEQ ID NO: 11) MBP 18 (ATGG)n6FAM- ATCCATTTACCTACCTGTTCATCC GGACCTCGTGAATTACAATCAC (SEQ ID NO: 14)T (SEQ ID NO: 13) D16S310 16 (ATAG)n VIC- AAAAAAGGACCTGCCTTTATCCGGGCAACAAGGAGAGACTCT (SEQ ID NO: 16) (SEQ ID NO: 15) THO1 11 (TCAT)nNED- TGTACACAGGGCTTCCGAGT AGGCTCTAGCAGCAGCTCAT (SEQ ID NO: 18)(SEQ ID NO: 17) IFN-A 9 (GT)n PET- GTAAGGTGGAAACCCCCACTTGCGCGTTAAGTTAATTGGTT (SEQ ID NO: 20) (SEQ ID NO: 19) MP3 D21S1245 21(AAAG)n VIC- TTGTTGAGGATTTTTGCATCA CCAGAAAATGACACATGAAGG (SEQ ID NO: 22)A (SEQ ID NO: 21) D20S48 20 (GT)n NED- TTGACCTGGATGAGCATGTGATGGTCTCCAGTCCCATCTG (SEQ ID NO: 24) (SEQ ID NO: 23) D9S171 9 (CA)n NED-GATCCTATTTTTCTTGGGGCTA TCTGTCTGCTGCCTCCTACA (SEQ ID NO: 26)(SEQ ID NO: 25) D16S476 16 (AAAG)n 6FAM- GGTGCTCTCTGCCCTATCTGGGCAACAAGAGCAAAACTCC (SEQ ID NO: 28) (SEQ ID NO: 27)

Amplified nucleic acids can be labeled in the present invention to helpdiscriminate one amplified product from another. In some aspects of thepresent invention, one of the primers of the pair is labeled with afluorescent dye. Table 1 identifies the fluorescent dyes (“6FAM,” “VIC,”“NED,” and “PET”) attached to the 5′ end of the forward primers. Thedyes' excitation maxima are distinct (520 nm, 554 nm, 575 nm, and 595nm, respectively), which allows discrimination between similarly sizedamplification products. A fluorescent dye, once attached to andoligonucleotide often exhibits a shift in its excitation maximum; butany excitation maximum referenced will the manufacturer's recitedexcitation maximum for the unconjugated dye. Other fluorescent dyes,capable of being attached to a nucleic acid primer, are contemplated inthe present invention. In some aspects, at least two of the fluorescentdyes used to label the primers have different maximum fluorescentemission wavelengths.

When fluorescently labeled primers are used to amplify microsatellitemarkers, one aspect of the invention is using genetic analysis devicecapable of detecting emitted fluorescence. In some aspects, the geneticanalysis device employs capillary electrophoresis to separate theamplification products and a CCD camera to detect emitted fluorescencefrom the products. For example, an ABI 3130 genetic analyzer utilizescapillary electrophoresis (CE) to separate amplification products bysize. This device also has a laser light source to excite thefluorescent dyes and a CCD camera to detect the fluorescently labeledamplification products. The output from a CE device is often in the formof an electropherogram, which is a graphical representation of the lightdetected during electrophoresis, thus allowing an observer to quicklyidentify the presence, absence, or variation in an expected peak.

FIG. 1 depicts an electropherogram of the IFN-A locus. The topelectropherogram is generated from a labeled amplification productoriginating from a buccal swab, while the bottom electropherogram isgenerated from a labeled amplification product originating from a urinesample. The buccal sample and the urine sample are from the samesubject, which allows determination of acquired LOH or MSI in thebladder sample. For example, the electropherograms in FIG. 1 show LOH inthe bladder sample. The last major peak in the buccal sample isapproximately the same size as the previous peak. These two peaksrepresent two alleles of the microsatellite locus. In the bladder sampleelectropherogram of FIG. 1, there is only one major peak, indicating LOHat that locus.

Software is also available that identifies STR alleles by using definedsignal collection bins. A “bin” corresponds to a particular region whereamplification products are expected to be detected. The bin in FIG. 1 isillustrated as a thin rectangle above the electropherogram and indicatesthat the expected amplification products will be between 132 and 152base pair range. The IFN-A alleles are the major peaks in the bin.However, for some microsatellites, alleles can be detected outside theassigned bin. For example, a homozygous allele in the D9S171microsatellite is detected outside the D9S171 bin in the bottomelectropherogram in FIG. 2.

FIG. 3 illustrates the presence of artifacts in the amplified product.This artifact is a minor peak outside the bin for the D16S476 locus.When analyzing samples for MSI or LOH in the D16S476 locus, thisartifact will not be considered as a separate allele. Artifacts of anamplification reaction will most often be present in the amplificationproducts for the bladder and the buccal swab samples. As shown in FIG.3, the artifact is constant in both samples.

EXAMPLES

The following examples are provided to further describe some of theembodiments disclosed herein. The examples are intended to illustrate,not to limit, the disclosed embodiments.

Example 1: Buccal Swab Extraction and Genomic DNA Extraction

Reagent preparation and buccal swab processing

a) Preparation Steps

Genomic DNA from buccal swabs was extracted using a commerciallyavailable protocol.

b) Methods for Avoiding Sample Cross-Contamination and Transferring ofSwab to Microfuge Tube

To reduce or eliminate the possibility of cross-sample contamination,only one tube was opened at a time when adding wash reagents and gloveswere changed if they came into contact with any sample liquid. Buccalswabs were placed into appropriately labeled 1.5 ml microcentrifugetubes, and the lids were closed onto the base of the tips where theshaft meets the tip. The shafts were broken off and discarded into abiohazard waste container, and the microfuge tube was closed. Swabshafts were cut off with scissors when necessary, but in those cases,the scissors were decontaminated with 10% NaClO (bleach) betweensamples.

c) DNA Extraction from Buccal Swab

400 μl of Phosphate-buffered saline (PBS) solution was added to eachsample tube. The tubes were vortexed vigorously for 50 seconds to assurethat the swabs were completely saturated. Tubes were inverted 10 timesto make sure PBS touched all areas of the tube. 20 μl of QiagenProteinase K stock solution and 400 μl of Buffer AL were added to eachsample tube, and the tubes were inverted 75 times. The samples werevortexed thoroughly for 50 seconds to assure proper lysis and incubatedat 56° C. for 60 minutes. The tubes were briefly centrifuged to removecondensation.

400 μl of 100% ethanol was added to each sample, and the tubes wereinverted 50 times or for 1 minute and then vortexed vigorously for 50seconds. The tubes were briefly centrifuged to remove condensation.

700 μl of lysate with ethanol were applied to the corresponding QIAampspin columns without wetting the rim, and the lids were closed. Touchingthe QIAamp membrane with the pipet tip was avoided. The tubes werecentrifuged at 8000 rpm for 1 minute, and the tubes containing thefiltrate were discarded. The QIAamp spin columns were placed into clean2 ml collection tubes. The nylon swabs were removed from the 1.5 mltubes and placed into the appropriate labeled spin columns. Whenextracting DNA from the cotton swabs, the swabs were rubbedperpendicularly to the rim of the column to loosen the cotton fibersfrom the wood so that the swabs could be removed from the sticks andleft in the columns. The tubes were centrifuged at 6000×g (8000 rpm) for1 min, and the tubes containing the filtrate were discarded.

500 μl of Buffer AW1 was added to each column without wetting the rim.The lids were closed, and the tubes were incubated at room temperaturefor 5 minutes. The tubes were centrifuged at 8000 rpm for 1 minute, andthe tubes containing the filtrate were discarded. The QIAamp spincolumns were placed into clean 2 ml collection tubes.

500 μl of Buffer AW2 was added to each column without wetting the rim.The lids were closed, and the tubes were centrifuged at 14000 rpm for 3minutes. The nylon/cotton swabs were removed from the columns. TheQIAamp spin columns were placed into new collection tubes andcentrifuged at 14000 rpm for 3 minutes. The tubes containing thefiltrate were discarded.

The QIAamp spin columns were placed into clean, labeled 1.5 mlcollection tubes, and the samples were incubated at 60° C. for 20-25minutes with the lids open. 50 μl of Buffer AE (pre-warmed to 65° C.)was added to each column, and the samples were incubated at roomtemperature for 5 minutes. The tubes were centrifuged at 8000 rpm toelute the DNA from the columns, and the samples were stored at 4° C.

Example 2: Bladder Sample DNA Extraction

a) DNA Extraction from Urine Sample

Bladder samples were obtained by collecting urine from the control andtest subjects. Urine samples were equilibrated to room temperature(15-25° C.). Samples were swirled, and a Hemastix strip was quicklydipped in the urine to test for the presence of blood. The results ofthe Hemastix test were read after one minute and recorded.

Urine samples were transferred to a labeled 50 mL conical tube andcentrifuged at 3500 rpm for 2 minutes. The supernatant was removed, andthe length of the pellet was measured from the base of the conical tube.If the pellet measured less than 2 mm in length from the base of theconical tube, 250 μL of DNA-grade water was added to suspend the pellet.If the pellet measured more than 2 mm in length, 1 ml of DNA-grade waterwas added to suspend the pellet.

The samples were vortexed, and 250 μL of each sample was aliquoted to a1.5 mL microcentrifuge tube. If there was more than 250 μL of sample,the extra sample was stored at −18±2° C. The total volume of urine andof water used to suspend the pellet was recorded, and the aliquotsamples were stored at −18±2° C. until they were ready for extraction.

Samples were thawed at room temperature, and 1120 μL Buffer AVL/CarrierRNA was added to each 250 μL aliquot. The samples were vortexed for 15seconds and incubated at room temperature (15-25° C.) for 10 minutes.

Each sample aliquot was split into 2 microcentrifuge tubes (685 μLeach), and 560 μL of 96-100% ethanol was added to each tube. Tubes werevortexed for 15 seconds to remove drops from inside the lid.

622 μL of the solution from the first microcentrifuge tube of eachsample was applied to the corresponding QIAamp spin column, the tubeswere centrifuged at >8000 rpm for 1 minute, and the filtrates werediscarded. The remaining 623 μL from the first microcentrifuge tube ofeach sample was added to the corresponding column, the tubes werecentrifuged at >8000 rpm for 1 minute, and the filtrates were discarded.622 μL of the solution from the second microcentrifuge tube of eachsample was applied to the corresponding QIAamp spin column, the tubeswere centrifuged at >8000 rpm for 1 minute, and the filtrates werediscarded. The remaining 623 μL from the second microcentrifuge tube ofeach sample was added to the same column, the tubes were centrifugedat >8000 rpm for 1 minute, and the filtrates were discarded.

500 μL Buffer AW1 was added to the spin columns, the tubes werecentrifuged at >8000 rpm for 1 minute, and the filtrates were discarded.

500 μL Buffer AW2 was added to the spin columns, the tubes werecentrifuged at full speed (≥14000 rpm) for 3 minutes, and the filtrateswere discarded. The tubes were centrifuged again at full speed for 1minute.

QIAamp spin columns were placed in microcentrifuge tubes, and 30 μLpre-heated (70° C.) DNA-grade water were added to the columns. Thesamples were incubated at room temperature (15-25° C.) for 5 minutes.The tubes were centrifuged at >8000 rpm for 2 minutes to elute the DNAfrom the columns, the eluate was dried in a speed vac on low heat for 20minutes of until the liquid had evaporated, and the DNA was dissolved in30 μL of DNA-grade water. The DNA samples were stored at −18° C.±2° C.for short term storage (up to six months) or ≤−65° C. indefinitely. Thework area was cleaned using 10% bleach, followed by deionized water.

b) Quantitation of Urine DNA

Quantifiler™ Human DNA Quantification Kits were used for thequantification of human genomic DNA. The kits are based on Taqmantechnology that uses two specific 5′ nuclease assays. One is a targetspecific assay for total human DNA or for the Y chromosome (male), andthe second is an internal PCR control (IPC), which detects inhibitiondue to too much DNA or common PCR inhibitors. The IPC components consistof an IPC template DNA, a synthetic sequence not found in nature, twoprimers, and one VIC-labeled probe. The target-specific assay componentsconsist of two primers to amplify the target DNA and a Taqman MGB probe.This contains the 6-carboxyfluorescein (FAM) reporter dye coupled to the5′ end and a minor groove binder (MGB) with a nonfluorescent quencher(NFQ) coupled to the 3′ end. The MGB increases the melting temperatureof the probe without increasing its length. The Quantifiler™ Human DNAQuantification primers and probe target the human telomerase reversetranscriptase gene (hTERT) located at chromosomal position 5p15.33; theamplicon length is 62 bases. The Quantifiler-Y™ quantification primerstarget the sex-determining region of the Y gene (SRY) at chromosomalposition Yp11.3; the amplicon length is 64 bases. During PCR, the TaqManprobe anneals to a specific region between the forward and reverseprimers. When the probe is intact, the quencher is close enough to thereporter molecule to suppress reporter fluorescence. As theDNA-Polymerase extends through the primer the reporter molecule iscleaved from the probe, causing increased fluorescence. The fluorescenceincrease is proportional to the amount of PCR product produced. For bothtotal and Y-specific DNA, the Quantifiler™ assay is used to quantifyunknown samples by interpolating their quantity from a standard curve.The assay can accurately quantify 63 pg to 100 ng of human DNA.

c) Preparation Steps

All racks, bench tops and equipment were decontaminated with 10% bleachand thoroughly dried. Instruments and pipettes were cleaned with 10%bleach, then water, then 100% methanol and dried thoroughly. It wasensured that all equipment meets the minimum standards for qualitycontrol, where appropriate. Any supplies or equipment taken from apost-amplification room to a pre-amplification room were sterilized with10% bleach before removal and again in the pre-amplification room priorto use.

To minimize the possibility of cross contamination between test andmatched control samples, reagents were never transferred from apost-amplification room to a pre-amplification room. No reagent aliquotswere returned to the original stock container.

The Quantifiler™ Human DNA standards were prepared fresh daily andstored at 5° C.±3° C. The Stock solution of DNA supplied withQuantifiler™ Kit is 200 ng/μL. The following quantities were used forthe creation of a standard curve: 50 ng/μL, 16.7 ng/μL, 5.56 ng/μL, 1.85ng/μL, 0.620 ng/μL, 0.210 ng/μL, 0.068 ng/μL and 0.023 ng/μL.

The appropriate amount of sterile water and Quantifiler™ Human DNAstandard was added to each tube, and the tubes were vortexed for 30seconds to mix thoroughly.

A negative (no template) control was prepared with 2 μL sterile water inplace of template DNA. The appropriate Primer Mix was removed from thefreezer, thawed, vortexed to mix and briefly centrifuge. Quantifiler™PCR Reaction Mix was removed from the refrigerator and pipetted up anddown to mix.

The PCR Master Mix was prepared as follows:

Reagent 1× Rxn Vol (*n) × Rxn Vol Quantifiler ™ Human Primer Mix 10.5(1.1 × n) × 10.5 μL Quantifiler ™ PCR Reaction Mix 12.5 (1.1 × n) × 12.5μL Total Volume 23 (1.1 × n) × 23 μL (*n) total number of reaction wellsand 1.1 is the 10% overage.

23 μL master mix was aliquoted into all the wells. After vortexing thesamples, 2 μL of each standard curve sample was added into theappropriate test wells of the 96 well plate. After all the standardcurve samples have been added, 2 μL of the negative (No TemplateControl-water) was added to the appropriate wells, and 2 μL of theunknown samples was added to the appropriate test wells. The plate wascovered with an adhesive tape, vortexed, and tapped to remove bubbles.Alternatively, the sample plate was centrifuged at 3000-4000 rpm for 2minutes to bring the contents to the bottom of the wells and to removeany bubbles. The plate was placed on the ABI Prism 7500 and 7500 FastReal Time PCR Systems for analysis.

d) Real Time PCR Analysis

The quantity of DNA present in each sample was determined by comparingits signal intensity to the intensity of the standard curve produced bythe human DNA standard controls and dividing that number by 2 to givethe ng/μl result. The standard curve results were examined to evaluatethe quality of the results from the quantification standard reactions.

The standard curve is a graph of the CT of quantification standardreactions plotted against the starting quantity of the standards. Thesoftware calculates the regression line by calculating the best fit withthe quantification standard data points. The R2 value is a measure ofthe closeness of fit between the standard curve regression line and theindividual CT data points of quantification standard reactions. A valueof 1.00 indicates a perfect fit between the regression line and the datapoints.

It was ensured that the R2 value was >0.98. If the R2 value was lessthan 0.98, the following were checked: 1) preparation of serialdilutions of quantification standards, 2) loading of reactions forquantification standards, and 3) failure of reactions containingquantification standards.

A slope close to −3.3 indicates optimal (100%) PCR amplificationefficiency. The typical slop range for the Quantifier Human kit is −2.9to −3.3, with an average of −3.1. If the standard curve did not complywith the guidelines, no more than two outlier data points could beeliminated by designating those wells as not in use. The wells in theplate document that corresponded to the outlier data points were removedand the plate was reanalyzed to incorporate the change. If theelimination of those data points did not improve the standard curve, thequantification was considered invalid and had to be repeated.

The positive control was ±10% of the actual concentration.

Example 3: Amplification of Microsatellite Loci

a) Optimal DNA Amount Used for MSA

The amount DNA extracted per urine sample dictated how the assay wouldbe performed. Optimally, a complete run using two multiplex PCRreactions was attempted. A concentration as low as 0.5 ng/μl in 15 μL ofwater could be used for testing of all multiplexes, using half thenominal concentration of DNA in each multiplex.

In cases where the total extracted DNA was less than 8 ng, the followingguidelines were used to proceed with the assay: if 2.00-3.99 ng DNA wasavailable from the urine sample, 2 ng DNA was used in each of PCRs 1, 2and 3; if 4.00-6.99 ng DNA was available from the urine sample, 3.5 ngDNA was used in each of PCRs 1, 2 and 3; if 7.00-8.99 ng DNA wasavailable from the urine sample, 4 ng DNA was used in each of PCRs 1, 2and 3.

The same amount of DNA was run for buccal sample analysis and urinesample analysis, regardless of whether there was more buccal DNA.

Patient paired buccal and urine DNA was PCR amplified simultaneously inthe same experiment, on the same thermal cycler, and under the sameconditions.

b) PCR for MSA: Primer Sequences

PCR Primer pairs for MSA assay are presented in Table 1. Threemultiplexes (MP1, MP2, MP3) were designed to amplify fourteen targets.

c) PCR for MSA: PCR Primer Master Mixes

For each primer assay master mix, each primer was diluted to a finalworking concentration of 2 pmol. The loci amplified in PCR1 were D4S243,FGA, D9S747, D17S654 and D17S695. The loci amplified in PCR2 wereD9S162, MBP, IFN-A, TH01 and D16S310. The loci amplified in PCR3 wereD21S1245, D20S48, D9S171 and D16S476.

For repeat singleplex reactions, primers were diluted to 5 μM bycombining 87.5 μL of DNA-grade water and 6.25 μL of forward and reverseprimers.

Primer assay master mixes were prepared in 100 μL for each primer mixand were scaled up to create more aliquots.

For PCR1, 145 μL of PCR grade water was added to a microcentrifuge tube.5 μL of Forward and Reverse primers for D9S747, D17S64, and D17S695 wereadded.

In separate microcentrifuge tubes, the Forward and Reverse primers forD4S243 were diluted 2-fold by adding 5 μL of primer to 5 μL of PCR gradewater, and 5 μL of the diluted Forward and Reverse primers were added tothe primer mix. 7.54 of Forward and Reverse primers for FGA were addedto the primer mix, and the primer master mix was thoroughly mixed.Single use 30 μL aliquots of the primer assay master mix were made.

For PCR2, 150 μL of PCR grade water was added to a microcentrifuge tube.5 μL of Forward and Reverse primers for D9S162, MBP, IFN-A were added.In separate microcentrifuge tubes, the Forward and Reverse primers forD16S310 and TH01 were diluted 2-fold by adding 5 μL of primer to 5 μL ofPCR grade water, and 5 μL of the diluted Forward and Reverse primerswere added to the primer mix, and the primer master mix was thoroughlymixed.

For PCR3, 150 μL of PCR grade water were added to a microcentrifugetube. 5 μL of Forward and Reverse primers for D21S1245 and D20S48 wereadded. In separate microcentrifuge tubes, the Forward and Reverseprimers for D9S171 and D16S476 were diluted 2-fold by adding 5 μL ofprimer to 5 μL of PCR grade water, and 5 μL of the diluted Forward andReverse primers were added to the primer mix, and the primer master mixwas thoroughly mixed.

d) PCR for MSA: PCR

Reagent volumes were multiplied by the number of samples plus positiveand negative controls to calculate the volumes needed for the mastermix. For every ten samples, 10% was added to account for volume lost inpipetting. Lot numbers were recorded on an MSA Amplification Worksheet.The following reagents were combined, in order, to create the reactionmixture for PCRs 1, 2 and 3: 12.5 μL 2× FastStart Taqman Probe Master, 1μL Primer Master Mix (1, 2 or 3) and 7.5 μL water, for a total of 21 μL.

21 μL master mix were aliquoted into each well for PCR1 and PCR2. Forcontrol reactions, 4 μL (multiplex 1, 2, 3) of control DNA were added tothe wells for the positive control and 4 (multiplex 1, 2, 3) of waterwere added to the wells for the negative control. For each patientsample reaction, 4 ng (4 μL) of DNA were added to PCR1, PCR2 and PCR3.For patient samples to be analyzed by singleplex PCR for repeatreactions, 1 ng (1 μL) of DNA was added to PCR1 and PCR2. PCR plates (orPCR tubes in 96 well PCR rack) were centrifuged briefly at 3000 rpm forabout 1 minute to bring all contents to the bottom of the plate.

The PCR conditions were as follows: one cycle of 95° C. for 11 minutes,32 cycles of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for 1minute, one cycle of 60° C. for 45 minutes, and a final hold at 4° C.Samples were then stored at −4° C. for short term storage or at −18° C.for long term storage. The PCR set-up area was cleaned with 10% bleach,followed by wiping with water and turning on the UV lamp for 5 minutes.

Example 4: Human DNA Identification with the ABI 3130 Genetic Analyzer

a) Sample Preparation of PCR Reaction for Genetic Analyzer

Sample loading buffer containing formamide (8.7 μL) and LIZ (0.3 sizestandard (9.0 μL total per well) was prepared. All unused wells in theplate column were filled with 10 μL formamide/LIZ master mix, and allwells in the adjacent even-numbered columns were filled with 10 μLformamide. 1.3 μL of each PCR reaction was added to the appropriate wellof the sample plate, and the plate was centrifuged briefly to bring thecontents to bottom of each well and remove bubbles.

The samples were heated at 95° C. for 3 minutes on a heat block and thenplaced on ice, at −18±2° C., on a cold block, or on a thermo cyclerblock at 4° C. for 2 minutes. The plate was assembled for the run byinserting septa in the sample plate, insert the samples into the platebase, and clamping on the plate retainer.

The Buffer Reservoir in position 1 was filled with fresh 1× GeneticAnalysis Buffer (1:10 dilution prepared from 10×3130 Buffer EDTA), andreservoir in positions 2-4 were filled with water.

The ABI 3130 Genetic Analyzers analyzes fragment data in sets of twocolumns descending for 16 wells per analytical folder. Patient pairedbuccal and urine samples were electrophoresed on the same run and in thesame folder of 16 well analyses.

a) Setting Up the Software for Genetic Analyzer

The software developed according to the invention, called PY software,is based on ABI 3130 Genetic Analyzer. It is operated by openingGeneMapperID ver 3.2 or higher, entering user ID (gmid) and password,selecting “Panel Manager” under the “Tools” button, selecting “NEW kit”under “File”, selecting “Microsatellite” and naming the panel “PY TestPanel”, clicking OK, placing the arrow on the new kit named “PY TestKit”, selecting “New Panel” under “File”, selecting “New Bins” under“Bins”, naming the new Binset, selecting “New Panel” under “File”,renaming Panel “Multiplex 1”, selecting Multiplex 1, and entering theinformation for each marker in the chart. The same steps were repeatedfor Multiplex 2 and Multiplex 3.

To create the Analysis Method, Genemapper manager is selected under“Tools”, the Analysis method editor is selected, and NEW is thenselected, the new Analysis Method is named “PY SOP” on the General tab,and “PY Test” is selected under Binset on the Allele tab. The RangerFiler button is selected, and the red channel is set to remove labelsfrom 186.5 to 188. The Peak Detector, Peak Quality, and Quality tabs areset.

The size standard is set by selecting Gene mapper manager under Tools,selecting the Size Standard Tab, selecting GS500LIZ standard and open.The values in the standard should be entered and saved as 100, 139, 150,160, 200, 300, 340, 350, 400, 450, 490, 500.

The instrument Protocol For 3130 analyzer is set by opening 3130 Datacollection software, selecting “Module Manager” and selecting “New”,selecting “FragmentAnalysis36_POP4:” as the template, creating a newname for Module, such as FragementAnalysis36_POP4_1.

The run settings are set as follows: Oven temperature 60° C., Pre runvoltage 15, Pre run time 180 seconds, Injection voltage 3.1 Kvolt,Injection time 8 second (or 5 seconds for re-injection of samples),Voltage number of steps 10NK, Voltage step interval 60 seconds, Datadelay time 1 second, Run voltage 15 volts, Run time 1500 sec.

Protocol Manager is selected, and “New” is selected, a name for theprotocol is created, such as “New Test Protocol”, the Type is selectedas “Regular”, the Run module is set to the module created above for the3130 analyzer, and the Dyeset is set to G5.

To prepare the Sample sheet, Applied Biosystems—Data Collection—Run DataCollector 2.0 is selected, the GA3130 folder is opened, “Plate Manager”is selected, option is selected, “New” is selected, and the name of thePlate is entered and recorded on the MSA PCR amplification worksheet.“GeneMapper-(Name of Computer)” is selected from Application and Ownername and Operator Name are filled in. On the GeneMapper plate editorscreen, the sample name is entered, priority is set to 100, sample typeis selected, size standard is set to Test Standard settings, either testpanel/multiplex 1 (PCR 1) or multiplex 2 (PCR 2) is selected, dependingon which multiplex is run, analysis method is set to “choose new test,”sections titled SNP are skipped, set user define 1, 2, and 3, Resultsgroup 1 is set to STR, Instrument protocol 1 is set to TEST PROTOCOL.

To perform the run, the folder entitled “3130” is opened, Run Scheduleris selected, “Find All” is selected, the run of interest is selected,the desired plate is highlighted, and the run is started. It tookapproximately 45 minutes to run 2 lanes, and when the runs werecomplete, the plates were removed, sealed with a Costar Thermowellsealer, and stored at −18±2° C. for up to 1 week.

4-C: Analyzing MSA Data with PY Software

The data was imported to GeneMapperID ver 3.2 or higher and analyzed bythe program. Manual editing of samples was carried out as necessary. Tomanually edit a sample whose SQ column has a red or yellow flag, sizematch editor (designated by size standard peaks) was selected, and thesizes of the peaks that were incorrect were manually changed by pressing“Override SQ” and “Apply”, and the samples were then re-analyzed.

c) Determining MSA Peak Sizes

Each analysis included data for paired patient samples of buccal DNA andurine DNA. These were viewed together. (i.e. 2 samples for PCR1, 2samples for PCR2 and 2 samples for PCR3). The 2 samples to be sized werehighlighted, and “Display Plots” was selected. The plots were reviewedby comparing the buccal sample to the urine sample for each multiplexreaction. Each channel (blue, red, yellow, green) was reviewed for eachsample and each multiplex. Allele-specific considerations for assigningpeaks was done. Examples of the patterns for IFN-α, D9S171 and D165476are shown in FIGS. 1-3. Upon completion of sizing, the Genotype Tab isselected, and “MSA ANALYSIS format is selected, and the genotypeinformation is used for statistical interpretation for acceptancecriteria.

d) Interpretation of Fragment Size Analysis on 3130 Genetic Analyzer

The analysis for each sample was performed by one analyst, and the dataentry was verified by a second analyst. The GeneMapper peak size andheight data table was accessed, and Acceptance Criteria was establishedusing more than 40 surgically resected bladder cancer samples and theircontrols and are shown in Table 2.

TABLE 2 MSA Markers Analyzed Repeat Color K562~allele Locus: Type: SizeRange: Channel: sizes: D4S243 (ATAG)n 165-192 bp Blue 169 bp FGA (TTTC)n299-361 bp Green 328 bp D9S747 (GATA)n 179-201 bp Green 185 bp D17S654(CA)n 194-218 bp Yellow 216 bp D9S162 (CA)n 117-148 bp Yellow 143 bpD17S695 (AAAG)n 170-220 bp Red 185, 200 bp MBP &A (ATGG)n200-242/119-151 Blue 207, 215, 119 bp D21S1245 (AAAG)n 209-293 bp Green236, 255 bp D16S310 (ATAG)n 127-170 bp Green 155, 160 bp D20S48 (GT)n251-269 bp Yellow 261 bp THO1 (TCAT)n 174-209 bp Yellow 198 bp D9S171(CA)n 109-129 bp Yellow 126 bp D16S476 (AAAG)n 176-230 bp Red 187 209 bpIFN-A (GT)n 132-152 bp Red no product

For samples to be called “normal”, the amplification products had tomatch the sizes (±1 bp) and colors indicated in Table 2 and have aminimum of 100 relative fluorescent units (RFU) for each heterozygousallele and a minimum of 200 RFU for homozygous alleles. The negativecontrol did not produce PCR products for any loci (RFU <100).

For samples to be called “cancer”, the amplification products had tomatch the sizes (±1 bp) and colors indicated in Table 2. For buccalsamples to be called “cancer”, the amplification products had to have aminimum of 100 relative fluorescent units (RFU) for each heterozygousallele and a minimum of 200 RFU for homozygous alleles. For urinesamples to be called “cancer”, the amplification products had to have aminimum of 100 relative fluorescent units (RFU) for at least one allele,and for two peaks to be considered alleles, the RFUs had to be within40% of one another. The maximum RFUs for an allele was 5000 RFU. Thecutoff values for each of the markers for determining LOH, based onpublished data and the analysis done here, are shown in Table 3.

TABLE 3 Cutoff Values by Marker (outside of range = LOH) Marker LowerLimit Upper Limit D4S243 0.68 1.33 FGA 0.60 1.43 D9S747 0.63 1.42D17S654 0.71 1.36 D17S695 0.49 1.61 MBP 0.63 1.45 MBPA 0.71 1.37 D16S3100.6 1.34 D9S162 0.51 1.53 THO1 0.46 1.53 IFN-A 0.68 1.43 D21S1245 0.581.42 D20S48 0.62 1.46 D9S171 0.72 1.36 D16S476 0.54 1.65e) Data Analysis from Buccal Swab Based MSA

Sample data were in ordered pairs for analysis. The descending order bysample was as follows: Buccal 1 with results for each locus in multiplex1, Urine 1 with results for each locus in multiplex 1, Buccal 1 withresults for each locus in multiplex 2, Urine 1 with results for eachlocus in multiplex 2, Buccal 3 with results for each locus in multiplex3, Urine 3 with results for each locus in multiplex 3. HL60 data wereorganized as follows: Multiplex 1, Multiplex 2 and Multiplex 3. Lociwere listed in the same order as those for the buccal. Column headingsof the spreadsheet were as follows: Sample ID, locus, allele 1 size,allele 2 size, two empty columns, allele 1 peak height, allele 2 peakheight. Data were imported into Excel for analysis.

The data was compared to the acceptance criteria above to determinewhether the data passed or failed the acceptance criteria. If a samplefailed the acceptance criteria, it was not used in the evaluation. Todetermine the status of a sample, the data was entered into the ExcelSpreadsheet “MSA Sample spreadsheet.xls”. This spreadsheet uses thefollowing calculation to determine the ratio of buccal to urine peakheight: Ratio=(urine 1 allele 1 peak height/urine 1 allele 2 peakheight)/buccal 1 allele 1 peak height/buccal 1 allele 2 peak height. Theratio and Negative/Positive for MSI or LOH marker status wereautomatically determined using the calculations based on the acceptancecriteria for each locus.

Loci demonstrating LOH (through loss of an allele) were consideredpositive for LOH, although ratios cannot be calculated for locidemonstrating complete loss. The ratio was only calculated forheterozygous loci and loci with complete data sets. The ratio wascompared with the cutoff data. Ratios falling outside the cutoff rangeswere considered positive for LOH. Ratios falling within the range weredesignated negative for MSI/LOH. If additional or shifted alleles weredetected in the urine, the loci were determined to be positive for MSIif the additional or shifted alleles were shifted ±3 bp within the bins,and the additional or shifted alleles had a minimum peak height of 100RFU.

f) Evaluation of Result from 3130 Genetic Analyzer: The Test RepeatCriteria

Repeat criteria were established for repeat testing when sufficient DNAremained to run the assay. If sufficient DNA was not present, theresults from the original run were considered valid for all loci meetingthe acceptance criteria for the assay and were reported. If an entireHL60 multiplex positive controls failed to meet the acceptance criteria,the sample data for the multiplex(es) that contained the failed lociwere not evaluated, and the appropriate HL60 and sample multiplexes wererepeated at the original DNA concentration. If one or more loci withinan HL60 control multiplex failed to meet the acceptance criteria, thesample data for the loci corresponding to the failed was not evaluated.The appropriate HL60 and sample multiplexes were repeated at theoriginal DNA concentration and only results for the loci that failedduring the first run were reported in the MSA Sample Spreadsheet. Datafrom loci that passed the acceptance criteria during the first run wereremoved from the sample sheet in the second run. If an entire samplemultiplex failed the acceptance criteria, the multiplex was repeatedusing the original concentration of DNA. If a sample had a single locuswith positive result, that locus was repeated in singleplex using 1 ngof DNA and 5 μM of each primer, and the results from both tests werereported. Loci containing additional urine peaks that indicatedmicrosatellite instability were repeated in singleplex to confirm theadditional peak, as described above. If any alleles within a locus had apeak height >5000 (off scale), loci were re-evaluated by re-injection ofthe multiplex with an injection time of 5 seconds.

A decision flow chart was used to determine which loci to include in there-injection evaluation. Homozygous alleles within a peak height >5000were not re-injected if they did not interfere with the ability toevaluate other loci. If re-injection values were used, both the buccaland urine values from the re-injected samples were reported in lieu ofthe buccal and urine values for the off-scale or obscured peaks obtainedin the first run. If still off scale or obscured, the result was NotEvaluable.

g) Final Reporting of Sample Results

Following all re-injection or repeat results, the final determination ofeach locus of a sample was reported on the original sample spreadsheetcontaining the multiplex data.

Example 5: Clinical Test—Detection of Bladder Cancer by MSA of UrinarySediment

a) Samples

The PY SOP and PY Test were used to predict the presence or lack ofbladder cancer in human biological samples. 5 matched buccal swabs andurine samples from “normal” patients and 5 matched buccal swabs andurine samples from bladder cancer patients were analyzed, along withpositive and negative DNA sample controls and, a no template negativecontrol. Each sample was identified with a unique specimen ID asassigned by the designated CLIA test lab (DCTL) sample accessioninggroup. All specimens were handled per CAP and CLIA regulations, insuringsample tracking throughout the testing process.

b) PCR for MSA

An MSA assay was carried out. The assay consisted of DNA extraction,normalization, and PCR amplification of STR markers of matched buccalswab and urine sediment genomic DNA. The PCR was done using primer setsthat flank the target STRs at 14 microsatellite loci. The 5′ end of eachprimer pair was fluorescently labeled to allow for detection of the PCRfragments by capillary electrophoresis. The primer pairs from Table 1,above, were used.

The PCR amplicons were resolved on a capillary-based gel electrophoresissystem that detects, sizes and determines the relative fluorescenceunits (RFU) for each fragment.

The RFUs of heterozygous alleles detected in the buccal swab DNA werecompared to the RFUs detected in the matched urine sample, and the ratioof RFUs from urine alleles to blood alleles was calculated. Markers thatexhibited values outside the ratios seen in normal samples were said toexhibit a loss of heterozygosity (LOH) that served as an indicator ofbladder cancer. See FIG. 4 for an example electropherogram.

c) MSA Assay Controls

Each sample and control was electrophoresed in the presence of a sizestandard that was used to calculate the fragment size of each PCRproduct. Positive Control Genomic DNA was from the American TissueCulture collection (ATCC) and from Applied Biosystems. The controls weretested at a concentration at the lower limit of detection as determinedin the clinical validation of the sample.

d) Instrument and Software

The instrument used for the assay was the 3130xl Genetic Analyzer fromApplied Biosystems, a 16 capillary instrument capable of generatinggenotyping data in 1 hour. The 3130xl Genetic analyzer handles a widevariety of gene detection assays, including DNA sequencing, SNPgenotyping, and fragment analysis. The 3130xl genetic analyzer operatesusing the Genemapper Software v2.0, which is 21 C.F.R. Part 11compliant. In accordance with CAP regulations, the instrument wascalibrated semi-annually with regard to spectral and spatialcalibration.

e) Sample Collection and Preparation

Two specimen types were collected: a buccal swab and urine sediment. Thebuccal swab was collected by a designated CLIA test lab (DCTL) using acollection kit and following clearly defined collection instructions.Briefly, (1) The buccal swab is stable for one week at 4° C.; (2) oncereceived at a DCTL, each case was accessioned and assigned a uniqueidentifier that followed the sample through the entire extractionprocess; (3) genomic DNA was extracted from the sample in CAP-accreditedlaboratories. The human DNA in the sample was quantified using theQuantifiler real-time PCR kit from Applied Biosystems, and the genomicDNA was normalized to 1 ng/μl.

Urine sediment was collected by a DCTL using a collection kit andfollowing clearly defined collection instructions. Briefly, (1) urinewas collected in a sterile collection cup, and a preservative was addedto stabilize the DNA in the urine. The urine and preservative weremixed, and the urine nucleic acids and proteins were preserved at roomtemperature for shipment. (2) Genomic DNA was purified form the urinesediment using the Urine DNA Isolation Maxi Kit from Norgen Biotech.This kit isolates both high molecular weight DNA (greater than 1 kb insize; mostly cell associated) and small molecular weight DNA (150-250bp; derived from the circulation) from 25 mL to 80 mL of urine. Thehuman DNA in the sample was quantified using the Quantifiler real-timePCR kit from Applied Biosystems, and the genomic DNA was normalized to 1ng/μl.

A set of matching urine and buccal swab specimens were obtained from 5patients diagnosed with bladder cancer and a set of matching sets from 5normal-healthy patients. The samples were obtained from BioreclaimationLLC. Two buccal swabs were collected from normal patients and one buccalswab was collected from cancer patients. Samples were de-identified,with any HIPPA sensitive information removed, by Bioreclaimation, and asample ID was assigned by Bioreclaimation for each specimen.

Biometric data for each specimen is provided in FIG. 5, and the samplenumbers are shown in Table 4.

TABLE 4 Sample numbers for analysis 5 Normal Swab and Urine MatchesUrine Lot # : 1st swab 2nd swab BRH769882 BRH769887A BRH769887BBRH769883 BRH769888A BRH769888B BRH769884 BRH769889A BRH769889BBRH769885 BRH769890A BRH769890B BRH769886 BRH769891A BRH769891B 5 CancerSwab and Urine Matches Urine Lot # : Swab BRH777321 BRH777326 BRH777322BRH777327 BRH777323 BRH777328 BRH777324 BRH777329 BRH777325 BRH777330f) DNA Extraction from Samples

The urine samples were centrifuged to pellet cells. The urine wasdecanted, and the pellets were resuspended in water. 250 μL of urinesediment slurry was pipetted into microtubes, and the remainder of theurine sediment was saved and stored at −20°±2° C. Buffer AVL/Carrier RNAwas added to each sample. Samples were vortexed and incubated at roomtemperature for 10 minutes. The QIA amp Viral RNA Mini Kit instructionswere followed for genomic DNA extraction.

g) DNA Quantitation and Normalization

Real Time PCR (TaqMan quantitative PCR) was used to determine theconcentration of human DNA in an extracted sample by direct assay of 1ng of DNA as determined by OD260 (see CBI DNASEQ00035). Human Beta Actingene is the target of the Taqman assay. A standard curve of human DNAstandard with a known concentration was used to compare results from thesample extract to determine concentration. TaqMan can determine evenslight differences in concentration and is sensitive down to 10 copiesof DNA molecules. Once the sample DNA concentration was determined, thesample was normalized to 1 ng/μl. The DNA concentrations in for thesamples are shown in Table 5.

TABLE 5 DNA concentrations of patient samples Quants Elution TotalSample ID's Test ID State (ng/μL) Volume (μl) Concentration (ng)BRH769883 PY MSA Urine-Normal 25.39 56.00 1421.84 BRH769882 PY MSAUrine-Normal 0.32 56.00 17.92 BRH769885 PY MSA Urine-Normal 7.00 56.00392.00 BRH769884 PY MSA Urine-Normal 1.38 56.00 77.28 BRH769886 PY MSAUrine-Normal 5.55 56.00 310.80 BRH769890A PY MSA Buccal-Normal 0.4046.00 18.40 BRH769890B PY MSA Buccal-Normal 1.71 46.00 78.66 BRH769888APY MSA Buccal-Normal 3.81 46.00 175.26 BRH769888B PY MSA Buccal-Normal6.32 46.00 290.72 BRH769889A PY MSA Buccal-Normal 15.14 46.00 696.44BRH769889B PY MSA Buccal-Normal 9.98 46.00 459.08 BRH769891A PY MSABuccal-Normal 0.33 46.00 15.18 BRH769891B PY MSA Buccal-Normal 4.1346.00 189.98 BRH769887A PY MSA Buccal-Normal 11.98 46.00 551.08BRH769887B PY MSA Buccal-Normal 13.89 46.00 638.94 BRH777326 PY MSABuccal-Cancer 16.05 46.00 738.30 BRH777327 PY MSA Buccal-Cancer 10.9646.00 504.16 BRH777328 PY MSA Buccal-Cancer 6.65 46.00 305.90 BRH777329PY MSA Buccal-Cancer 4.91 46.00 225.86 BRH777330 PY MSA Buccal-Cancer41.22 46.00 1896.12 BRH777321 PY MSA Urine-Cancer 1.00 56.00 56.00BRH777322 PY MSA Urine-Cancer 26.57 56.00 1487.92 BRH777323 PY MSAUrine-Cancer 5.40 56.00 302.40 BRH777324 PY MSA Urine-Cancer 37.75 56.002114.00 BRH777325 PY MSA Urine-Cancer 279.21 56.00 15635.76h) MSA PCR and Analysis

The MSA PCRs were carried out as described in Example 3 using Amplitaqgold as the enzyme. The Acceptance Criteria described in Example 3 andshown in Table 2 were used. The analysis was carried out as described inExample 4, and the cutoff values presented in Table 3 were used todetect LOH and MSI. The criteria presented in Example 4f were used toclassify LOH or MSI.

i) Results

Table 6 provides the Sample Pair IDs and their corresponding urine andbuccal swab Sample IDs provided by Bioreclamation.

TABLE 6 Sample Pair IDs and corresponding Sample IDs from BioReclamationLLC Sample Pair ID Bioreclamation ID Matched sets Normal Urine Lot # :1st swab 2nd swab N1 BRH769882 BRH769887A BRH769887B N2 BRH769883BRH769888A BRH769888B N3 BRH769884 BRH769889A BRH769889B N4 BRH769885BRH769890A BRH769890B N5 BRH769886 BRH769891A BRH769891B 5 Cancer Swaband Urine Matches Bladder Cancer Urine Lot # : Swab C1 BRH777321BRH777326 C2 BRH777322 BRH777327 C3 BRH777323 BRH777328 C4 BRH777324BRH777329 C5 BRH777325 BRH777330

FIGS. 6a-6e , respectively show the raw MSA data for the Sample Pair IDsN1-N5 as defined in Table 6. FIGS. 6f-6j , respectively show the raw MSAdata for the Sample Pair IDs C1-C5 as defined in Table 6.

Table 7 shows the results of the MSA carried out, with “N”=normal (noLOH), “LOH”=loss of heterozygosity between a paired buccal and urinesample, and “MSI”=microsatellite instability observed in a paired buccaland urine sample. “Final call” is considered “N” if fewer than two lociexhibited LOH or MSI, or “C” for cancerous if two or more loci exhibitedLOH or MSI.

TABLE 7 Summary of MSA results MSA results Buccal/Urine Pairs SamplePair ID Loci N1 N2 N3 N4 N5 C1 C2 C3 C4 C5 MSA D4- NI N N N N N N N NI NS243 Panel FGA N N N N N N N N LOH N D9- N N N N NI N NI NI LOH M- S747SI D17- N N N NI N N NI N N N S654 D17- N N N NE NI NI N NI N N S695 MBPN N N N IN LOH N LOH NI L- OH MBPA N N NI N N N NI NI N N D16- N N N NNI NI N N N N S310 D9- NI NI N NI N N N Ni LOH N S162 THO1 NI NI N N N NN LOH LOH N IFN-A NI NI N N N N N LOH N NI D21- NI N NE N N N MSI NI LOHL- S1245 OH D20- N N NI N N LOH LOH N N N S48 D9- N N N N NI N NI NI N NS171 D16- NI N N N N N NI NI NI N S476 Final N N N N N C C C C C Call NI= NON-INFORMATIVE (HOMOZYGOUS) NE = NON-EVALUABLE (AMPLIFICATION FAILED)LOH = LOSS OF HETEROZYGOSITY N = Normal MSI = microsattelite instablityC = CancerIn summary, samples clinically diagnosed and confirmed by cystoscopywere determined to positive for the disease by the MSA methods presentedhere. Each of the normal controls were negative for MSI and LOH for allof the 14 markers tested. For the bladder cancer samples, at least twoof eight particular markers were positive for LOH. The eight markerswere FGA, D98747, MBP, D98162, TH01, IFN-A, D21S1245 and D20S48.Therefore, the buccal-based MSA analysis presented here requires the useof only eight markers to accurately detect bladder cancer.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is, therefore, intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the

The disclosures of each patent, patent application, and publicationcited or described in this document are hereby incorporated herein byreference, in its entirety.

What is claimed:
 1. A method for analyzing heterozygosity inmicrosatellite loci from a bladder sample and a matched control buccalsample from a human subject, the method comprising: a) using a pluralityof pairs of primers to amplify a set of microsatellite markers from thebladder sample and the buccal matched control sample to produce a set ofamplification products, wherein the set of microsatellite markerscomprises FGA, D9S747, MBP, D9S162, THO1, IFN-A, D21S1245, and D20S48,and wherein at least one of the primers of each pair of primers islabeled with a fluorescent dye to provide relative fluorescent units ofeach amplification product; and, b) calculating ratios of RFUs of eachamplification product from the bladder sample to RFUs of eachamplification product from the buccal matched control sample, wherein acalculated ratio falling outside a cutoff range of a ratio of anamplification product from a normal bladder sample to an amplificationproduct from a buccal matched control indicates loss of heterozygosity(LOH) in the microsatellite locus, wherein: (1) a primer consisting ofSEQ ID NO: 3 and an optional fluorescent dye label and (2) a primerconsisting of SEQ ID NO: 4 and an optional fluorescent dye label are thepair of primers for FGA; (3) a primer consisting of SEQ ID NO: 5 and anoptional fluorescent dye label and (4) a primer consisting of SEQ ID NO:6 and an optional fluorescent dye label are the pair of primers forD9S747; (5) a primer consisting of SEQ ID NO: 13 and an optionalfluorescent dye label and (6) a primer consisting of SEQ ID NO: 14 andan optional fluorescent dye label are the pair of primers for MBP; (7) aprimer consisting of SEQ ID NO: 11 and an optional fluorescent dye labeland (8) a primer consisting of SEQ ID NO: 12 and an optional fluorescentdye label are the pair of primers for D9S162; (9) a primer consisting ofSEQ ID NO: 17 and an optional fluorescent dye label and (10) a primerconsisting of SEQ ID NO: 18 and an optional fluorescent dye label arethe pair of primers for THO1; (11) a primer consisting of SEQ ID NO: 19and an optional fluorescent dye label and (12) a primer consisting ofSEQ ID NO: 20 and an optional fluorescent dye label are the pair ofprimers for IFN-A; (13) a primer consisting of SEQ ID NO: 21 and anoptional fluorescent dye label and (14) a primer consisting of SEQ IDNO: 22 and an optional fluorescent dye label are the pair of primers forD21S1245; and/or (15) a primer consisting of SEQ ID NO: 23 and anoptional fluorescent dye label and (16) a primer consisting of SEQ IDNO: 24 and an optional fluorescent dye label are the pair of primers forD20S48.
 2. The method of claim 1, wherein amplification of the set ofmicrosatellite markers comprises at least two multiplex amplificationreactions.
 3. The method of claim 2, wherein the amplification of theset of microsatellite markers comprises three multiplex amplificationreactions, and wherein each multiplex amplification reaction comprises adifferent combination of markers from the set of microsatellite markers.4. The method of claim 3, wherein templates for the first multiplexreaction amplification comprise markers FGA and D9S747.
 5. The method ofclaim 3, wherein templates for the second multiplex reactionamplification comprise markers D9S162, MBP, IFN-A, and THO1.
 6. Themethod of claim 3, wherein templates for the third multiplex reactionamplification comprise markers D21S1245 and D20S48.
 7. The method ofclaim 1, wherein at least two of the fluorescent dyes used to label theprimers have different maximum fluorescent emission wavelengths.
 8. Themethod of claim 1, wherein the bladder sample is obtained from a urinesample, or is a bladder biopsy sample.
 9. The method of claim 1, whereinthe bladder sample is a bladder biopsy sample.
 10. The method of claim1, wherein the matched control buccal sample is buccal cells.
 11. Themethod of claim 1, wherein the set of microsatellite markers furthercomprises D4S243, D17S654, D17S695, D16S310, D9S171, and D16S476. 12.The method of claim 11, wherein the amplification of the set of markerscomprises three multiplex amplification reactions, and wherein eachmultiplex amplification reaction comprises a different combination ofmarkers from the set of microsatellite markers.
 13. The method of claim12, wherein a first multiplex reaction amplifies markers D4S243, FGA,D9S747, D17S654, and D17S695.
 14. The method of claim 12, wherein asecond multiplex reaction amplifies markers D9162, MBP, IFN-A, THO1, andD16S310.
 15. The method of claim 12, wherein a third multiplex reactionamplifies markers D21S1245, D20S48, D9S171, and D16S476.
 16. The methodof claim 1, wherein the upper and lower limits of the cutoff ranges ofthe ratios of the RFU of each amplification product from the normalbladder sample to the RFU of each amplification product from the buccalmatched control sample are as follows: Marker Lower Limit Upper LimitFGA 0.60 1.43 D9S747 0.63 1.42 MBP 0.63 1.45 D9S162 0.51 1.53 THO1 0.461.53 IFN-A 0.68 1.43 D21S1245 0.58 1.42 D20S48 0.62  1.46.


17. The method of claim 1, wherein the primers consisting of SEQ ID NO:3, 5, 11, 13, 17, 19, 21, and 23 are labeled with a fluorescent dye. 18.The method of claim 17, wherein the fluorescent dye is VIC, NED, 6FAM,or PET.
 19. The method of claim 17, wherein the fluorescent dye isattached to the 5′ end of the primer.
 20. The method of claim 18,wherein: the 5′ end of the primers consisting of SEQ ID NO: 3, 5, and 21are labelled with VIC; the 5′ end of the primers consisting of SEQ IDNO: 11, 17, 23 are labelled with NED; the 5′ end of the primerconsisting of SEQ ID NO: 13 is labelled with 6-FAM; and/or the 5′ end ofthe primer consisting of SEQ ID NO: 19 is labelled with PET.
 21. Themethod of claim 1, wherein: a primer consisting of SEQ ID NO: 3 and afluorescent dye label and a primer consisting of SEQ ID NO: 4 are thepair of primers for FGA; a primer consisting of SEQ ID NO: 5 and afluorescent dye label and a primer consisting of SEQ ID NO: 6 are thepair of primers for D9S747; a primer consisting of SEQ ID NO: 13 and afluorescent dye label and a primer consisting of SEQ ID NO: 14 are thepair of primers for MBP; a primer consisting of SEQ ID NO: 11 and afluorescent dye label and a primer consisting of SEQ ID NO: 12 are thepair of primers for D9S162; a primer consisting of SEQ ID NO: 17 and afluorescent dye label and a primer consisting of SEQ ID NO: 18 are thepair of primers for THO1; a primer consisting of SEQ ID NO: 19 and afluorescent dye label and a primer consisting of SEQ ID NO: 20 are thepair of primers for IFN-A; a primer consisting of SEQ ID NO: 21 and afluorescent dye label and a primer consisting of SEQ ID NO: 22 are thepair of primers for D21S1245; and/or a primer consisting of SEQ ID NO:23 and a fluorescent dye label and a primer consisting of SEQ ID NO: 24are the pair of primers for D20S48.
 22. The method of claim 21, wherein:the 5′ end of the primers having a nucleotide sequence consisting of SEQID NO: 3, 5, and 21 are labelled with VIC; the 5′ end of the primershaving a nucleotide sequence consisting of SEQ ID NO: 11, 17, 23 arelabelled with NED; the 5′ end of the primer having a nucleotide sequenceconsisting of SEQ ID NO: 13 is labelled with 6-FAM; and/or the 5′ end ofthe primer having a nucleotide sequence consisting of SEQ ID NO: 19 islabelled with PET.
 23. The method of claim 2, wherein the amplificationof the set of microsatellite markers comprises three multiplexamplification reactions.
 24. The method of claim 11, wherein theamplification of the set of markers comprises three multiplexamplification reactions.